Characterization of Antioxidant Agent Producing Bacteria form Intestinal Flora f Farm Animals

Abstract

Excessive reactive oxygen species (ROS) damage cell membrane, cellular components and cause oxidative damage. They also lead to various human disorders such as diabetes, cancer, Alzheimer, rheumatoid arthritis and inflammation. The body possesses antioxidative defense and repair mechanisms to protect against such damages. But sometime this system is unable to prevent entire damage caused by ROS. In such conditions, supplementation of exogenous antioxidant has considered better options. Several synthetic antioxidants (butylated hydroxy-toluene (BHT), propyl-gallate and butylated hydroxyl-anisole (BHA) are extensively used, but their safety is questioned due to recent reports on their toxicity. In this scenario, exploration of natural and safer antioxidant from bio-resources and their replacement with synthetic antioxidant is highly commended. Lactic acid bacteria (LAB) are an emerging natural source of cellular antioxidants. LAB are diversely dispersed in nature. Previously LAB isolate from dairy products and some from human fecal matter. Information about few isolated strains of LAB is available from farm animals. The aims of this work were (a) to explore lactic acid bacteria from farm animals for their antioxidant potential. b) to check probiotic potential of strains with promises antioxidant capability and identify potential competent strain (c) to investigate in vivo antioxidant ability of competent strain (d) to perform its safety assessment and finally e) the partially purify secondary metabolite responsible for the antioxidant property of the strain. The strains were identified using conventional microbiological and molecular methods. The antioxidant scavenging assay against three radicals (DPPH, superoxide anion and OH radical) and inhibition of lipid peroxidation were used to evaluate the antioxidant potential in vitro. Twenty three strains were investigated in intact cells, the supernatant and cell lysate. Highest antioxidant activity was noticed in intact cell and supernatant while weak activity was observed in cell lysate. The promiser antioxidant activity in four assays was recorded. Seven out of twenty three LAB strains displayed above 60% antioxidant activity in all four tests and were selected for further study. The probiotic potential of the selected seven strains was assessed through tolerance against different pH and temp, resistance to bile salt, sodium chloride salt and simulated gastric intestinal environment. The strains were able to tolerate temperature (30-55C), pH (2-10), bile salt (up to 2%), sodium chloride (up to 8%) and simulated gastrointestinal environment (≥ 80% survival) proving their probiotic nature. In addition the strains were noticed to pose least threat to eukaryotic cells identified by very weak hemolytic activity (0.2-1.2%) against host erythrocytes. The seven strains were identified as Enterococcus rati (An8), Lactobacillus brevis (An13, An15 An26, An27 and An28) and Pediococcus acidilactici (An17) on the basis of 16S rRNA gene sequencing. They were awarded accession no are MF183967, MH185807, MH185808, MG882401, MG882402 and MG000874 from NCBI Gene bank. These strains are also deposited in Fungal Culture Bank for the use of other researchers. However, among all the performance of An28 was excellent. Therefore, this strain was chosen for in vivo validation of its antioxidant potential. The strain An28 was identified as L. brevis MG000874. The Efficacy of this strain was checked by analyzing selected oxidative stress parameters (SOD, CAT, GST and GSH) and general health in d-galactose model of oxidative stress. Two experiments were performed in this regard. A pilot study was conducted to determine the dose of d-galactose required to induce oxidative stress in mice. Dose of 150mg/Kg BW/day for sixty days could successfully induced oxidative stress. The oxidative stress was identified by depletion in SOD, CAT and GSH and elevation of GST elevation for eight weeks. In the main trial, the prophylactic effect of L. brevis MG000874 supplementation worked out. Physical sign and symptoms as well as estimation of oxidative stress indicators of liver, kidney and serum were measured. The analysis of SOD, CAT, GST and GSH provided evidence that L. brevis MG000874 can resist the occurrence of oxidative stress. The continue feeding of L. brevis MG000874 @109cfu/ml for a period of 8 weeks did not adversely affect the general health of animals. This strain was fed to the Mus musculus for eight weeks. After eight weeks of treatment, liver and kidney homogenate were used for analysis of known markers of toxicity, including ALT, AST, ALP, T. bilirubin, Albumin and protein from liver and Urea and Creatinine test from kidney. In addition histological studies on liver and kidney were also carried out on the size of central vein, sinusoids and hepatocytes of liver and glomerulus, corpuscles, PCT and DCT in kidney were taken into account. LFT and RFT analysis revealed the safe nature of L. brevis MG000874 which was also supported by histological findings. In the end the metabolites released in the cell free supernatant by L. brevis MG000874 were partially purified. In this procedure, the metabolites having antioxidant property were separated through thin layer chromatography (TLC). These separated antioxidant components were analyzed in High performance liquid chromatography (HPLC) and Attenuated Total Reflectance Infrared (ATR-IR) for determination of purity and the chemical nature of pure compound. The analysis revealed that the component was aldehyde in nature. It is concluded that L. brevis MG000874 produce antioxidative compounds of aldehyde nature that may serve as a novel agent for controlling the oxidative stress in host. Live L. brevis MG000874 or its metabolites may be included in the list of potential probiotics and prebiotics respectively.